Development of a cyclic adenosine monophosphate assay for Gi-coupled G protein-coupled receptors by utilizing the endogenous calcitonin activity in Chinese hamster ovary cells.

Activation of G(i)-coupled G protein-coupled receptor (GPCRs) by their ligands leads to inhibition of adenylyl cyclase (AC) and reduction of cyclic adenosine monophosphate (cAMP) levels in cells. The traditional cAMP assay for G(i)-coupled GPCRs commonly uses forskolin, a nonspecific AC activator, to increase the basal cAMP level in cells to create an assay window for ligand detection. However, there is still a need to develop a nonforskolin-based cAMP assay because of the challenges inherent in titrating the concentration of forskolin to achieve a reliable assay window, along with issues related to the cAMP-independent effects of forskolin. Herein, we describe such an assay by utilizing the endogenous activity of the calcitonin receptor in Chinese hamster ovary (CHO) cells. The calcitonin receptor is a G(s)-coupled GPCR that, when activated by calcitonin, leads to the stimulation of AC and increases cAMP in cells. Thus, we use calcitonin, instead of forskolin, to increase the basal cAMP level in CHO cells to achieve an assay window. We demonstrated that calcitonin peptides robustly increased cAMP accumulation in several CHO cell lines stably expressing well-known G(i)-coupled GPCRs, such as the Dopamine D2 receptor, the Opioid μ receptor, or the Cannabinoid receptor-1. Agonists of these G(i)-coupled GPCRs attenuated calcitonin-induced cAMP production in their receptor stable cell lines. On the other hand, antagonists and/or inverse agonists blocked the effects of their agonists on calcitonin-induced cAMP production. This calcitonin-based cAMP assay has been demonstrated to be sensitive and robust and exhibited acceptable assay windows (signal/noise ratio) and, thus, can be applied to screen for agonists and antagonists/inverse agonists of G(i)-coupled GPCRs in high-throughput screening formats.